1. Field of the Invention
This invention relates to novel compositions useful for detecting visually the presence of hydrogen peroxide and/or substances having peroxidative activity and particularly to compositions useful for detecting low levels of hydrogen peroxide.
The detection and quantitative determination of hydrogen peroxide and compounds yielding hydrogen peroxide are of importance in many areas, for example, in the detection of hydrogen peroxide produced in the enzymatic assay of substances such as glucose, cholesterol, uric acid, etc. by the activity of enzymes such as glucose oxidase, cholesterol oxidase, uricase, etc. in the presence of oxygen. The quantity of enzyme substrate present in a sample is determinable from the amount of hydrogen peroxide produced and detected.
Known compositions for detecting and/or quantifying hydrogen peroxide in such systems generally comprise a substance having perioxidative activity, e.g. peroxidase and peroxidase-like substances, and material which undergoes a detectable change (generally a color change) in the presence of hydrogen peroxide and the peroxidative substance. A complete list of the prior art which describes such compositions is too extensive for presentation here. However, a few representative patents which describe such materials are: U.S. Pat. Nos. 2,912,309, 2,981,606, 3,349,006, 3,092,465, 3,558,435, 3,595,755, 3,627,697, 3,627,698, 3,630,847, 3,654,179, 3,654,180 and 3,853,470. Examples of various color forming substrates of peroxidase and peroxidase-like substances which have been suggested in the prior art include, among others, the following substances with a coupler where necessary:
(1) Monoamines, such as aniline and its derivatives, ortho-toluidine, para-toluidine, etc.;
(2) Diamines, such as ortho-phenylenediamine, N,N'-dimethyl-para-phenylenediamine, N,N'-diethyl phenylenediamine, benzidine (which produces a blue or brown color), dianisidine (turns green or brown), etc.;
(3) Phenols, such as phenol per se (producing a yellow color), thymol, ortho-, meta- and para-cresols (producing a green-yellowing color, a pink color and a milky suspension, respectively), alpha-naphthol (producing a magenta color), betanaphthol (producing a white precipitate), etc.;
(4) Polyphenols, such as catechol, guaiacol (which forms an orange color), orcinol, pyrogallol (producing a reddish or yellow color), p,p-dihydroxydiphenyl and phloroglycinol;
(5) Aromatic acids, such as solicyclic, pyrocatechuic and gallic acids;
(6) Leuco dyes, such as leucomalachite green (to produce malachite green) and leucophenolphthalein (desirably employed in an alkaline medium);
(7) Colored dyes, such as 2,6-dichlorophenolindophenol;
(8) Various biological substances, such as epinephrine, the flavones, tyrosine, dihydroxyphenylalanine (producing an orange-reddish color) and tryptophane;
(9) Other substances, such as gum guaiac, guaiaconic acid, potassium, sodium, and other water soluble iodides; and bilirubin (producing a greenish color); and
(10) Such particular dyes as 2,2'-azine -di(3-ethylbenzothiazoline-(6)-sulfonic acid) and 3,3'-diaminobenzidine.
Although the above mentioned substances are in general useful as indicator systems for the detection of hydrogen peroxide, there are instances when the concentration of hydrogen peroxide to be analyzed is too low to produce sufficient detectable color from such indicators either due to the source of the hydrogen peroxide, the necessity for dilution, or the overall detection method. A specific example of such a concentration problem occurs in the detection of hydrogen peroxide produced from the low levels of uric acid present in blood serum (1-15 mg/dl). Where the environment of use permits compensation for this shortcoming of prior art compositions it is usually overcome by the measurement of relatively large volumes of the detectable product, e.g., by increasing the diameter of the cuvette in a solution assay for uric acid so as to additively increase the relative density of the color produced.
All analytical techniques do not, however, permit the use of such modifications to increase the effective density of indicator produced. In other applications the amount of indicator produced is small, due to either the initially very low concentration of analyte to be assayed using the hydrogen peroxide quantifying system or the necessity for high dilution levels to obtain proper dissolution or the like, that such manipulative approaches are not practical.
Such problems are particularly acute when the analyte determination is performed in a multilayer element of the type described in Belgian Pat. No. 801,742 issued Jan. 2, 1974 in the names of Przybylowicz and Millikan, and most acute when an analyte of low concentration, such as uric acid, is being assayed in such an element. In these cases, using the relatively thin indicator or reagent layers (on the order of less than a mil) that are desirable in such elements, the density of the color formed can be rather low. Increasing the thickness of the color-providing layer to provide greater density may be undesirable, as it can increase reaction times, create problems in layer preparation, etc. Consequently, other techniques for increasing the effective density of dye produced in the indicator reaction had to be found, if elements of this type were to be used reliably for the assay of low concentration blood serum components such as uric acid. The methods and compositions described herein are of course equally useful in the assay of materials and analytes, and specifically serum components, other than uric acid and in other media than multilayer elements and are particularly useful in assays requiring the detection of hydrogen peroxide where large dilutions of analyte solution are required for one reason or another.